Endocrinol Metab. 25(2):103-109, June 2010 ORIGINAL ARTICLE 지방세포에서비중을이용한지방유래줄기세포가풍부한분획의분리방법 김민경 박용순 박희순 1 최정묵 1 김원준 2 박세은 2 이은정 2 박철영 2 이원영 2 오기원 2 박성우 2 김선우 2 서광식 3 우정택 3 한양대학교생활과학대학식품영양학과, 강북삼성병원당뇨병연구소 1, 성균관대학교의과대학강북삼성병원내분비내과 2, 경희대학교의과대학내분비대사내과 3 Isolation of Density Enrichment Fraction of Adipose-Derived Stem Cells from Stromal Vascular Fraction by Gradient Centrifugation Method Min Kyung Kim, Yong Soon Park, Hee Soon Park 1, Jung Mook Choi 1, Won Jun Kim 2, Se Eun Park 2, Eun Jung Rhee 2, Cheol-Young Park 2, Won Young Lee 2, Ki Won Oh 2, Sung Woo Park 2, Sun Woo Kim 2, Kwang Sik Suh 3, Jeong Taek Woo 3 Department of Food and Nutrition, Hanyang University, Seoul, Korea; Diabetes Research Institute 1, Kangbuk Samsung Medical Center, Seoul, Korea; Department of Internal Medicine 2, Kangbuk Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; Department of Endocrinology-Metabolism 3, College of Medicine, Kyunghee University, Seoul, Korea Background: Adipose tissues include multipotent cells, the same as bone marrow-derived mesenchymal stem cells. The stromal vascular fractions (SVFs) from adipose tissues represent a heterogeneous cell population. The purpose of this study was to isolate and purify adipose-derived stem cells (ASCs) in SVFs by the density gradient method. Methods: SVFs were extracted from the subcutaneous, epididymal, mesenteric and retroperitoneal adipose tissue of 8 weeks old male Sprague-Dawley rats (n = 15) and these were separated into 4 layers according to a Nycodenz gradient (: < 11%, : 11-13%, : 13-19% and : 19-30%). The post-confluent SVFs were cultured in adipogenic medium for 2 days, in insulin medium for 2 days and in 10% fetal bovine serum medium for 5 days. To observe lipid droplets in SVFs, we performed Oil Red O staining. Results: The SVFs cellular fractions (,, and ) were isolated by density gradient centrifugation from the adipose tissues of rats. The SVFs extracted to fraction 3 () had the most abundant cells compared to that of the other fractions. However fraction 1 () or 2 () had a superior ability to make lipid droplets. The adipogenic differentiation of or 2 was higher than that of the unfractionated cells. The SVFs extracted from retroperitoneal adipose tissue had the highest efficiency for adipogenic differentiation, whereas the SVFs from mesenteric adipose tissue did not differentiate. Conclusion: This density gradient fractionated method leads to efficient isolation and purification of cells with the characteristics of ASCs. (Endocrinol Metab 25:103-109, 2010) Key Words: Rat adipose tissue, Adipose-derived stem cell, Stromal vascular fraction, Density gradient, Nycodenz 서론 줄기세포 (stem cell) 는적절한신호에의한자기복제및다양한조직으로분화할수있는능력을가진전구세포로서발생단계에서부터인체의장기를형성하고성장후에는장기및조직의기능을 Received: 12 October 2009, Accepted: 23 December 2009 Corresponding author: Cheol-Young Park Department of Internal Medicine, Kangbuk Samsung Medical Center, 108 Pyeong-dong, Jongno-gu, Seoul 110-746, Korea Tel: +82.2-2001-2440, Fax: +82.2-2001-1588, E-mail: cydoctor@chol.com 복원하는데중요한역할을한다. 줄기세포는발생초기배반포 (blastocyst) 에서얻어지는배아줄기세포 (embryonic stem cell) 와발생과정이끝난성체또는태반에서얻어지는성체줄기세포 (adult stem cell) 가있다 [1]. 이두가지줄기세포는서로다른특징을가지고있는데, 배아줄기세포는미분화상태에서자가증식능력이뛰어나지만분화잠재성을알기어려워생체내이식을한경우불필요한세포의증식등암발생가능성을고려해야한다. 또한배아줄기세포의이용은생명체이용이라는점에서많은윤리적인문제를안고있어실질적인사용에제한이따른다. 반면, 성체줄기세포는생체내
104 Kim MK, et al. 에이식된후장기특성에맞게분화하는특이성및본래의세포특성과는다른종류의세포로교차분화할수있는유연성을가지고있고, 다양한세포로분화될수있는다잠재성이있음이밝혀지면서성체줄기세포를통한세포치료의가능성은높아지고있다 [2]. 성체줄기세포중중간엽줄기세포를얻기위한연구들은주로골수에서이루어져왔으며, 골수줄기세포를이용하여다양한조직으로의분화등많은연구가이루어져왔다 [3-7]. 그러나골수에서의세포획득은환자의고통을수반하며임상에적용하기위한충분한양의세포를얻기위해서는여러번채취해야하는부담이존재한다 [8,9]. 골수와같은간엽에서유래하며다양한기질세포들을포함하고있는지방조직은또다른줄기세포의원천으로 Zuk 등 [10,11] 은지방추출물안에줄기세포로추정되는세포들이있으며, 이를지방유래줄기세포 (adipose-derived stem cells, ASCs) 라고명명하였다. 지방조직은많은양의조직채취가용이하여줄기세포를수확하는데좋은조건을가지고있으며, ASCs는배양시안정적인성장과증식을보여주고분화를유도하였을때골수줄기세포와같이다양한세포로의분화가가능하다는것이증명되었다 [12]. 현재 ASCs는지방조직의성숙지방세포, 적혈구등을제거한나머지기질세포 (stromal vascular fraction, SVF) 를분리배양하여얻는다 [13]. 그러나이 SVF에는지방전구세포, 지방모세포등의지방세포의각분화단계에따른여러가지성분들이포함되어있다. 세포막표면에존재하는단백질에특이적인항체를이용하여분리하는방법 [10,11] 이개발되어있지만고비용으로인하여제한적이고대중적으로사용하기어려운단점이있다. 인간과동물의장기및조직에서생존율과순도가높은세포를분리하기위한방법으로세포의크기와밀도에기초한방법들이활용 [14] 되고있는데밀도차등 (density gradient) 원심분리는죽은세포와뒤섞인상태로있는살아있는세포 (live cell) 를분리해낼수있는것으로알려져있으며, 주로간세포의분리에이용되고있다 [15]. 밀도차등원심분리에사용되는매질로는 Percoll, metrizamide, Ficoll 400, Nycodenz, stractan 등이있는데, 비이온성요오드화합물인 Nycodenz 는세포독성, 삼투압손상및응집반응이적다고알려져있다 [14-16]. 이에본연구에서는지방조직의 SVF에서분리한세포를표면항원분석의비용적인측면을고려한새로운방법인 Nycodenz 를이용한밀도차등원심분리를이용하여생존율과순도가높은분획및지방세포로분화되는적정구획을찾아보고자하였다. 재료및방법 1. Stromal vascular fraction의분리 8주령의수컷 Sprague-Dawley 흰쥐 (n = 15, 242.7 ± 1.3 g) 를사용하여 subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT), mesenteric adipose tissue (MAT), retroperitoneal adipose tissue (RAT) 를채취하였다. 각각의지방조직을 2% bovine serum albumin (BSA, Gibco, USA) 을넣은 Hank s Balanced Salt Solution (HBSS, Gibco, USA) 으로세척하여작은조각으로분쇄하였다. 분쇄시킨조직을 0.075% collagenase Type II (Sigma-Aldrich, USA) 로 37 에서 30분처리한다음, 10% fetal bovine serum (FBS), 0.2% Fungizone, 1% Penicillin Streptomycin (Gibco, USA) 을함유한 Dulbecco s Modified Eagle Medium (DMEM, Gibco, USA) 으로효소반응을정지시키고 100 μm 나일론여과지 (mesh filter, BD bioscience, USA) 에통과시켜불필요한조직을제거하였다. 200 g에서 10분동안원심분리하여상층에부유된지질과지방세포층을제거한다음하부의모아진세포를 RBC lysis buffer (ebioscience, San Diego, CA, USA) 에 10분간처리하고여과및원심분리하여 SVF를모은다. 2. 비중에따른세포의분리 30% wt/vol 위에 19%, 13%, 11% 의농도별비연속 Nycodenz (Sigma, USA) buffer를만든다음지방조직의 SVF를 11% Nycodenz buffer에넣어 250 g에서 30분동안원심분리하였다. 상층액과각농도별경계면 (interface) 에서수집한 SVF를각각 (< 11% Nycodenz), (11-13% Nycodenz interface), (13-19% Nycodenz interface), (19-30% Nycodenz interface) 로명명하였다. 3. 지방세포로의분화유도지방조직별로수집한각분획의 SVF는 10% FBS, 0.2% Fungizone, 1% Penicillin Streptomycin을함유한 DMEM 배지 (Complement medium, CM) 에희석하여 12 well plate에 1 10 5 cell/ml를넣어 37, 5% CO 2 배양기에서배양하였다. 초기 24시간후에부착되지않은세포는 phosphate buffered saline (PBS, Gibco, USA) 으로세척하여제거한후 2-3일간격으로새로운배지를교환하면서포화상태에이를때까지배양하였다. 지방세포로의분화유도를위해배양액에 520 μm 3-isobutyl-methylxanthine (IBMX), 1 μm dexamethasone, 1 mg/ml insulin을첨가한지방세포분화유도배지 (MDI medium, Adipogenic medium) 를 2일간처리한다음 1 mg/ ml insulin만을첨가한배지 (Insulin medium) 로다시 2일간처리하였다. CM으로 6일동안배양하면서지방방울 (lipid vacuole) 의형성여부를현미경으로관찰하였다. 4. 분화도측정지방세포의확인은세포내지방의축적을알수있는 Oil Red O stain을이용하였다. 세포를 10% formaldehyde 로 30분고정한후에 60% isopropanol로세척한다음 60% isopropanol에 Oil Red O (Sigma-Aldrich, USA) 를녹인 Oil red O solution 으로 30분이상염색하였다. 염색한세포는현미경으로관찰하였으며, 100% isopropa-
Isolation of Density Enrichment Fraction of Adipose-Derived Stem Cells from Stromal Vascular Fraction by Gradient Centrifugation Method 105 nol 로 Oil red O de-stain 을하여 ELISA Reader (Model 680 Microplate Reader, Bio-Rad, USA) 로 490 nm 에서흡광도를측정하였다. 1. 지방조직의분포 결과 각지방조직의분포는 SAT 가전체지방의 50.2% 로양이가장많 고, EAT 22.8%, MAT 15.8%, RAT 11.2% 를차지했으며전체지방량 은평균 4.7 ± 0.2 g 였다 (Fig. 1). 다른농도의 Nycodenz buffer 를이 용하여원심분리를한결과밀도에따라 4 개의분획에서 SVF 를얻 을수있었다. 2. 지방조직별지방줄기세포의양과분화정도 Trypan blue 염색법으로지방조직별로살아있는세포의수를측 정한결과, MAT 로부터분리된세포 (M-SVF) 의수가가장많았으며, E-SVF, S-SVF, R-SVF 순서였다 (Fig. 2A). S-SVF 와 R-SVF 는 post confluence 에도달하는속도가빨랐으며, M-SVF 와 E-SVF 는상대적으 % ATs of body weight 2.5 2.0 1.5 1.0 0.5 0.0 Percentage of ATs ATs SAT EAT MAT RAT Fig. 1. Amount of adipose tissues is most extensive in subcutaneous, epididymal, mesenteric, and lowest in retroperitoneal adipose tissue. Data is expressed as mean ± SE. 로느린속도를보였다. 7일동안지방세포로분화시킨결과는세포수를측정한결과와는반대로 R-SVF의분화가가장잘이루어졌고, S-SVF도높은분화율을보였으나 E-SVF는상대적으로분화가잘되지않았고 M-SVF는분화가거의이루어지지않았다. 분화된지방세포의 Lipid vacuoles 에서도지방조직에따른차이를관찰하였다. 양은많지만크기가작은 lipid vacuole을보였던 S-SVF에비해 E-SVF는상대적으로양이적었지만커다란 lipid vacuole을형성하고있었다. R-SVF는지방세포의양이가장많았으며 lipid vacuoles 또한큰편이었다 (Fig. 3). 3. 분획에따른지방조직별기질세포의양밀도차등원심분리를이용하여나눈지방조직의분획별생세포수는부위에따른 4 종류의지방조직에서비슷한패턴을보였다. 세포수가가장적은분획은 이고, 의세포수는가장많았다 (Fig. 2B). 차이점은 RAT로부터분리된세포는 가 보다많았지만다른지방조직은 가 보다세포수가적었다. CM으로 2 일정도배양했을때관찰한세포의모양은유래된지방조직및분획에상관없이모두섬유아세포 (fibroblast) 의형태로차이가없었다. 4. 분획에따른지방조직별지방줄기세포의분화정도분획을나누지않은 SVF를대조군 (con) 으로하여분획을나눈 SVF를지방세포로 7일동안분화시켜현미경과 Oil red O stain으로확인하였다. 분화 7일째되었을때분획에따라 lipid vacuoles 의크기와양이달랐다. S-SVF와 R-SVF는모든분획에서전체적으로지방세포를고르게형성하였다 (Fig. 4). 그러나 E-SVF는부분적으로분화가이루어졌으며분획에따라지방세포의양에차이가있음을현미경으로관찰하였고, M-SVF는지방세포를거의찾아볼수없었다. 축적된지방을 Oil red O stain을하여현미경으로분화정도를확인하였을때, 분획마다고른분화를보였던 S-SVF와 R-SVF는대조군과의차이를확인하기어려웠으나상대적으로분화율이떨어지 % cell of ATs weight 100 80 60 40 20 The number of cells in ATs % cell of ATs weight 50 40 30 20 10 The number of cell fractions 0 0 SAT EAT MAT RAT SAT EAT MAT RAT A B Fig. 2. Cell numbers of undifferentiated SVFs were estimated by trypan blue staining. A. Mesenteric adipose tissue had the most abundant cells than other adipose depots (MAT > EAT > RAT > SAT). B. SVFs extracted to fraction 3 () had highest cell numbers than other fractions.
Kim MK, et al. 106 M-SVF R-SVF S-SVF E-SVF M-SVF R-SVF Control E-SVF Control S-SVF Fig. 3. Post-confluent ASCs were cultured in adipogenic medium. On day 7 of adipogenic induction, lipid vacuoles were observed within the ASCs, and a significant difference was detected in the number, size and distribution patterns of the lipid vacuoles (magnification, 200). Fig. 4. Comparisons in the differentiation were examined by Oil red O stain for adipogenesis. The retroperitoneal and subcutaneous adipose tissue derived stem cells had the highest efficiency in adipogenic differentiation whereas the mesenteric adipose tissue dereived stem cells did not differentiate (magnification, 200). 3.5 (Fig. 4). Oil red O de-stain으로 분화율을 정량분석하여 지방조직 3.0 마다 분획별로 분화 양상을 비교하였다. S-SVF와 R-SVF는 의 2.5 분화율이 가장 우수한 것으로 나타났으며 S-SVF와 R-SVF보다 각각 2배, 1.5배 유의하게 높은 분화 효율을 보였다 (Fig. 5). E-SVF와 M-SVF는 의 분화율이 가장 높았지만 E-SVF는 S-SVF, R-SVF에 비해서 분화율이 전체적으로 크게 떨어지는 수준이었고, M-SVF는 거의 분화가 되지 않았다(Fig. 5). 고 찰 본 연구는 지방조직으로부터 SVF만을 추출하던 기존의 방식에 기술적인 부분을 보강하여 SD rat에서 채취한 4부위의 지방조직에 Oil red O de-stain 는 E-SVF는 대조군에 비해 분획을 나누었을 때 분화가 잘 되었다 The mean level of adipogenic differentiation control 2.0 1.5 1.0 0.5 0.0 S-SVF E-SVF M-SVF R-SVF Fig. 5. After Oil red O staining, the optical density of Oil red O-positive cells was assessed by ELISA reader. The retroperitoneal adipose tissue derived stem cells had the highest efficiency in adipogenic differentiation. The adipogenic differentiation of fraction 1 or 2 was higher compared to that of the unfractionated cells (control) and other fractions. Data are expressed as mean ± SE. 서 SVF를 분리한 뒤 Nycodenz로 밀도차등원심분리를 하여 생존율 과 순도가 높은 지방세포로 분화되는 적정 분획을 찾았다. Ny- 세포, 내피세포, 면역세포, 지방전구세포 등 여러 세포가 혼합되어 codenz를 비롯하여 밀도차등원심분리에 이용되는 매질들은 현재 있다[20-23]. 골수 및 조직으로부터 추출된 중간엽줄기세포(mesen- 주로 간세포의 분리에 이용되고 있으며[14,17], Nycodenz를 이용한 chymal stem cell, MSC)는 일반적인 배양 상태에서 세포부착능력을 밀도차등원심분리는 세포에 대한 안정성이 높고 경제적 측면에서 가지며, in vitro에서 다분화능을 보이고, 특유의 표면 항원 발현이 손쉽게 이용할 수 있는 방법이다[18,19]. 라는 세 가지 특징을 가지고 있다[24]. MSC는 형광물질이나 자석이 ASCs를 얻기 위해 현재까지 이루어지고 있는 방법은 1960년대 붙은 항체를 유세포분석기(FACS) 혹은 자기활성세포분석기(MACS) Rodbell에 의해 시작된 방법으로 성숙한 지방세포와 혈구 성분만을 로 줄기세포임을 확인한다. 일반적으로 MSC에 사용되는 음성항체 제거한 SVF를 분리하여 배양하기 때문에 섬유아세포, 혈관평활근 는 조혈줄기세포 및 과립구 계통의 항원인 CD34, CD45, CD14 (또는
Isolation of Density Enrichment Fraction of Adipose-Derived Stem Cells from Stromal Vascular Fraction by Gradient Centrifugation Method 107 CD11b), CD79α ( 또는 CD19), HLA-DR이며, 양성항체는 CD105, CD73, CD90이다 [25]. SVF는줄기세포항원인 CD166, CD44, CD29, CD73, CD90, CD105 를가지고있으며이들항원은 passage가증가함에따라증가하고 CD11, CD14, CD45, CD34는 passage가증가할수록발현이감소되거나없어진다 [26-28]. 그러나사람과동물의 ASC에서 CD90, CD34, CD106, CD105, Stro-1은초기 passage에서더잘나타나는등발현이가변적이다. ASC와 bone marrow mesenchymal stem cell (BSC) 는비슷한항원을가지고있지만이들사이의표면항원의구분이명확하지않다 [29,30]. 또한항체를이용한확인에서도상반된결과를보이는경우들이있는데 Zuk 등 [10] 의연구에서분리된기질세포에서는조혈계표면항원인 CD34의비율이매우낮았다. Planat-Benard 등 [31] 은 CD34의비율을 90% 이상이라고보고했으며 Miranville 등 [32] 은부위별로조금씩다르지만약 30-60% 이상차지하였다고보고하였다. 이는순수한지방줄기세포를분리하는데있어기술적인어려움이존재하며표면항원분석은단지줄기세포의기원을확인해주는것에지나지않음을뜻한다. 그러나본연구에서는밀도차등원심분리를이용하여얻어진 SVF의표면항원분석을시행하지않음으로써지방조직별, 분획별차이를규명하는데한계가있으므로이를보완하기위한향후추가연구가필요할것으로사료된다. 8주령수컷 SD rat으로부터채취한지방조직은 SAT가 50% 이상을차지하였고, EAT, MAT, RAT 순이었다. 그러나각지방조직으로부터추출한 SVF의세포수는 MAT에서가장많았으며, 그다음이 EAT, SAT, RAT 순인것으로보아지방량과 SVF의세포수가비례하는것이아님을알수있었다. 특히, 50% 이상을차지했던 SAT의경우 SVF의세포수가적다는것은 SAT 안에성숙한지방세포가많음을의미하며반대로 MAT는조직량이적은만큼성숙한지방세포도적었기때문에 SVF의세포수가많았던것으로보인다. 한편, Nycodenz 를이용한밀도차등원심분리로확보된 SVF는 4 부위지방조직에서모두 3번째분획이가장많았다. 그러나지방세포로의분화가가장잘되는분획은어떤부위의지방조직이냐에따라다르지만첫번째또는두번째분획에서분화가가장잘이루어졌다. 즉, 분화가가장잘이루어진분획은지방세포로분화할수있는줄기세포를가장 homogeneous 하게가지고있으며, 밀도차등원심분리를통해가장많은세포가모였던세번째분획은지방줄기세포로써의 heterogeneous 함을의미한다. Density gradient인 percoll 을이용하여 rat의 inguinal tissue의 SVF를분리한연구에서도첫번째 (1.018-1.033), 두번째 (1.033-1.049) 분획이 homogeneous 하며, 세번째 (1.049-1.062) 분획은형태상으로도 heterogeneous 하다고보고하였다 [33]. 또한분획을나누지않은 SVF와비교했을때분획을나눈 SVF의분화결과를비교해보면첫번째또는두번째분획이분획을나누지않은 SVF보다분화효율이우수하였다. 따라서밀도차등원심분리가지방조직으로부터줄기세포를수득하는데있어보다 순도가높은 SVF를얻을수있게해준다는것을증명하였다. 지방조직에따라분화율을비교해보면 R-SVF가가장분화가잘되었고그다음이 S-SVF였으며, E-SVF는상대적으로분화율이떨어지고 M-SVF는분화가거의이루어지지않았다. MAT로부터분리한 SVF의세포수가가장많았던점을고려하면 MAT에는줄기세포보다는내피세포, 섬유아세포등과같은다른세포들을더많이가지고있음을추측할수있다. 반면가장세포수가적었던 RAT는분화효율이가장좋았던점을미루어 ASCs가많으며, SAT도마찬가지로다른조직에비해 ASCs가많다는것을알수있다. 최근에는 ASC를지방세포뿐만아니라근육세포, 연골세포, 골세포, 신경세포등으로분화를시키는연구들이이루어지고있다 [10, 11,34,35]. 본연구는지방세포로의분화효율이우수한분획을찾았으나다른세포로의분화에대한시도는이루어지지않은제한점이있다. 향후지방조직으로부터분리한 SVF의밀도차등원심분리를통해얻은각분획을지방세포로의분화뿐만아니라골세포, 연골세포, 근육세포로의분화를유도하여분화효율이높은분획을찾고자하는추가적인연구가필요할것으로사료된다. 요약배경 : 지방조직은골수유래의중간엽줄기세포와같은다분화능력을가진성체줄기세포를포함한다. 지방조직으로부터추출한지방유래줄기세포가다양한분화유도를통해지방세포, 골세포, 근육세포로분화할수있음이밝혀졌다. 본연구에서는지방조직으로부터얻은기질세포에서지방세포로의탁월한분화능력을가지고있는순수한지방유래줄기세포를추출해낼수있는지알아보았다. 방법 : 수컷 Sprague-Dawley 쥐 (n = 15) 의 4부위지방조직 ( 피하지방, 부고환지방, 장간막지방, 후복막지방 ) 에서불필요한조직을제거한나머지기질세포만을얻는다. Nycodenz 를이용하여기질세포를밀도차등원심분리하여 4개의분획을얻어각각을배양시킨다. 포화상태에이른지방줄기세포를지방세포로분화를유도하여세포내지방축적여부를염색을통해확인한다. 결과 : 밀도차등원심분리로지방조직기질세포는 4개의분획 (Fx- 1,,, ) 으로나뉘었으며, 4부위지방조직모두세번째분획 () 의세포수가가장많았다. 그러나지방세포로의분화는 4부위지방조직모두두번째분획 () 에서탁월했으며분획을나누지않은것 (control) 과비교했을때더나은분화능력을보였다. 지방으로분화가가장잘되는지방조직은 retroperitoneal fat tissue 였으며그다음이 subcutaneous fat tissue였고, epididymal fat tissue 는상대적으로분화율이떨어지는것을관찰하였다. Mesenteric fat tissue는분화가거의되지않았다. 결론 : 지방조직별, 밀도별로지방조직기질세포를나누어분리함으로써지방조직으로의분화능력이우수한지방줄기세포를찾을
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